Arabidopsis Transcriptomics Reveals the Role of Lipoxygenase2 (AtLOX2) in Wound-Induced Responses

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

In arabidopsis, jasmonate signaling leads increased foliar levels of glucosinolates (GSLs), S-and N-rich specialized metabolites that contribute protection against insects and pathogens [18][19][20].In the leaves, GSLs are spatially separated from their activating enzyme, myrosinase [21].Herbivore damage disrupting foliar tissue allows contact between the myrosinases and GSLs, resulting in the hydrolysis of the thioester bond and releasing an unstable compound that undergoes rearrangement to produce toxic compounds such as isothiocyanates, thiocyanates and nitriles [18].Though both aliphatic and indolic GSLs may negatively affect insect herbivores, in general, aliphatic GSLs have a stronger negative impact on generalist caterpillar herbivores while indolic GSLs negatively affect aphids [22][23][24][25][26].
Given the increase in jasmonates within seconds after wounding [27,28], jasmonate biosynthetic enzymes are thought to be constitutively present and regulated both transcriptionally and post-translationally in response to stress [29,30].Indeed, an enzyme that catalyzes an early step in jasmonate biosynthesis, AtLOX2, is constitutively phosphorylated at Ser 600 and dephosphorylated in wounded plants [31].In vitro studies using phosphovariants confirmed that phosphorylation of AtLOX2 at Ser 600 may affect enzyme activity with lower activity observed in the phosphomimics (Ser 600 -to-Asp, Ser 600 -to-Met) compared with the unphosphorylated WT (Ser 600 ) or phosphonull variants (Ser 600 -to-Ala) [32].This is likely due to the fact that Ser 600 phosphorylation interferes with the positioning of the substrate on the enzyme active site.
13S-LOXs, such as AtLOX2, catalyze the addition of oxygen to the 13-position of a chloroplast membrane-derived polyunsaturated fatty acid, typically α-linolenic acid.In arabidopsis, there are four functional 13S-LOXs, AtLOX2, AtLOX3, AtLOX4 and AtLOX6, that contribute to the jasmonate burst.A hierarchy is noted where LOX2 and LOX6 activity results in jasmonate production that leads to the expression of AtLOX2, AtLOX3 and AtLOX4 [33].Thus, AtLOX2 contributes to the early foliar jasmonate burst, as does AtLOX6, and is primarily associated with mesophyll and bundle sheath cells and important in local signaling [27,28,33].In contrast, AtLOX6 is localized near the vasculature and likely plays an important role in long-range jasmonate signals.AtLOX3 and AtLOX4 are associated with the xylem and phloem, respectively, and are responsible for later (4 h after wounding) jasmonate biosynthesis, along with AtLOX2.
Even though these four functional AtLOXs contribute to jasmonate biosynthesis in response to plant stress, AtLOX2 is thought to be the most important for plant resistance against caterpillar herbivory [28,33].Generalist caterpillars of the Egyptian cotton leafworm, Spodoptera littoralis, gained nearly twice the biomass when reared on the lox2 mutant compared with WT or lox3/4/6 plants [33].As well, LOX2 activity results in singlet oxygen ( 1 O 2 ) production in wounded leaf tissue, which may affect transcriptional responses [34,35].
In this study, we seek to dissect the role of AtLOX2 in plant defense further by comparing wound-induced responses after foliar wounding of Col-0 and the loss-of-function lox2 mutant.We combined bioinformatic analyses of transcriptomics data with phytohormone and GSL analyses focusing on phytohormone signaling and 1 O 2 -responsive gene expression as well as GSL metabolism.Our results show that constitutive AtLOX2-associated jasmonate biosynthesis may be involved in antagonizing salicylic acid (SA) levels.In wounded arabidopsis, AtLOX2 exerts a high degree of control on the plants' response to 3 of 31 damage.In particular, the mechanically damage-associated jasmonate burst was lower in the lox2 mutant, which is reflected in the reduced expression of late wound-induced genes.

Results
Arabidopsis has four functional 13S-LOXs involved in the biosynthesis of jasmonates in response to wounding or biotic stresses, such as insect herbivory or pathogens, making it complex to tease out the role of individual enzymes in plant resistance [36].In wounded arabidopsis leaves, these proteins work in a hierarchical arrangement with AtLOX2, primarily contributing to plant resistance against caterpillar herbivory [33].Using a lox2 mutant [28], we conducted a time course transcriptomic experiment to understand the role of AtLOX2 in the plant response to wounding, particularly in connection to plant protection.

Transcriptome Profiling and Mapping onto the Arabidopsis Genome
After processing to remove the adaptor and poor-quality reads, RNA-Seq produced an average of 39.7 million 100 bp reads per sample, all with a Pfred score at or above 20.The high-quality reads were aligned with the arabidopsis TAIR10 genome with an average mapping efficiency of 97.8% uniquely mapped reads (Supplemental Table S4).

Constitutive Phytohormone and Gene Expression Levels
Constitutively, gene expression was highly conserved between the two genotypes (~20,469 genes) (Figure 1A); less than 1% were differentially expressed between WT and lox2 at any time point.In WT plants compared with lox2 mutants, there was an increase in specialized metabolism, glutathione metabolism and phenylpropanoid biosynthesis (Figure 1B,C).However, some pathogen resistance-related genes had higher expression levels in lox2 plants compared with WT plants, which may reflect the increased SA levels in these plants (Figure 2).These transcripts included genes that encode enzymes in SA biosynthesis (i.e., AtICS1, AtCM3, AtAIM1 [37]) and SA-responsive genes (i.e., AtPR1, AtNIMIN1, AtNIMIN2, AtGRXS13 [38]).In contrast, constitutive levels of the jasmonate phytohormone OPDA and JA were higher in WT plants compared with the lox2 mutant (Figure 3A

Arabidopsis Responses to Foliar Damage
After wounding, the levels of JA and JA-Ile rose in damaged rosette leaves in both or only WT genotypes, respectively (Figure 3B,C).As expected, levels of these phytohormones were ~7.5 times higher in WT than the lox2 mutant.Jasmonate signaling is dynamic with the early expression of genes encoding biosynthetic enzymes and positive regulators (Figure 3D) [39][40][41].In addition, the jasmonate burst is tempered either by catabolizing jasmonates to a less active form or by the activity of JAZ proteins that bind to and repress MYC2/3/4 transcription factors [42,43].
linolenic and linoleic acid biosynthesis, phytohormone and MAPK signaling, terpenoid biosynthesis and wound healing (cutin, suberin and wax biosynthesis) as well as redox metabolism (glutathione metabolism) was observed (Figure 4A,B,E,F).Though most of the processes observed 1 h post-damage were also observed later, there was also an increase in primary metabolic pathways 12 h after wounding.In contrast, though the same general pathways were upregulated in the lox2 mutant (Figure 4C,D), fewer genes were strongly expressed (Figure 4G,H).In general, WT and lox2 plants showed similar wound-induced responses (Figure 5A,D).One hour post-damage, out of the 249 wound-induced genes observed in WT plants, 81% were also induced in damaged lox2 (Figure 5A,B).A stronger difference in woundinduced gene expression between WT and lox2 plants was observed 12 h after wounding (Figure 5D,E).At this time point, only 76 wound-induced genes were identified in WT and shared 31% gene expression with damaged lox2.Based on this, wound-induced gene expression patterns were divided into six distinct groups as follows: Pattern 1-general early (1 h) wound-induced gene expression (Figure 5A), Pattern 2-early (1 h) wound-induced gene expression with higher expression in WT plants (Figure 5B), Pattern 3-early (1 h) wound-induced gene expression with higher expression in lox2 plants (Figure 5C), Pattern 4-general late (12 h) wound-induced gene expression (Figure 5D), Pattern 5-late (12 h) wound-induced gene expression with higher expression in WT plants (Figure 5E) and Pattern 6-late (12 h) wound-induced gene expression with higher expression in lox2 plants (Figure 5F).
Gene expression associated with other phytohormones and their signaling pathways that may be involved in crosstalk were also observed.Wound-induced expression of AtGA2OX6 and AtGA2OX8, encoding enzymes that oxidize gibberellins lowering their availability [59], as well as the negative growth regulator DELLA protein AtRGAL3 [60,61], may reflect the shift from growth to defense [62].Even though abscisic acid (ABA) levels were not affected by wounding (Supplemental Figure S1), a number of ABA-related genes were induced 1 h after wounding.The expression of the ABA receptor AtPYL6 and ABA-responsive genes (i.e., AtRAS1, AtERD7, AtOSCA1.4,AtERD10, AtCOR78 [63][64][65][66][67]) were upregulated early after foliar damage.ABA-related responses help minimize water loss from damaged leaves as well as enhance plant defense responses [68][69][70].In response to wounding, synergistic defensive responses between jasmonates and ethylene are often observed [71]; genes encoding ethylene biosynthetic enzymes (i.e., AtCSP2 and AtACS8) and ethylene responses (i.e., AtERF2 and AtRAP2.6)were noted in early wound-induced responses.

Pattern 6: Late Wound-Induced Gene Expression in lox2 Plants
Only a few genes showed lox2-specific late wound-induced expression (Figure 5F, Supplemental Table S5F).Expression of the SA-responsive gene AtPRLIP2 was observed in wounded lox2 mutants [92,93].

Glucosinolates
Constitutively or 12 h post-wounding, the foliar GSL profile did not differ between the two genotypes, reflecting the gene expression profile of GSL biosynthetic enzymes (Figure 7A).However, genes encoding GSL transcriptional regulators and biosynthetic enzymes show a strong diurnal cycle with stronger expression in the light phase, particularly for genes involved in aliphatic GSL biosynthesis (Figure 7B).Genes encoding indole GSL methyltransferase1 (AtIGMT1), the GSL transporter AtNPF2.10 and lectin JAL23 (a polymerization factor and putative activator of the myrosinase PYK10 [94]) showed strong wound-induction in WT plants.

Pattern 6: Late Wound-Induced Gene Expression in lox2 Plants
Only a few genes showed lox2-specific late wound-induced expression (Figure 5F, Supplemental Table S5F).Expression of the SA-responsive gene AtPRLIP2 was observed in wounded lox2 mutants [92,93].

Glucosinolates
Constitutively or 12 h post-wounding, the foliar GSL profile did not differ between the two genotypes, reflecting the gene expression profile of GSL biosynthetic enzymes (Figure 7A).However, genes encoding GSL transcriptional regulators and biosynthetic enzymes show a strong diurnal cycle with stronger expression in the light phase, particularly for genes involved in aliphatic GSL biosynthesis (Figure 7B).Genes encoding indole GSL methyltransferase1 (AtIGMT1), the GSL transporter AtNPF2.10 and lectin JAL23 (a polymerization factor and putative activator of the myrosinase PYK10 [94]) showed strong wound-induction in WT plants.

Discussion
Our results highlight the transcriptional dynamics of wound-induced jasmonate biosynthesis and the importance of AtLOX2 in sustained jasmonate signaling leading to plant resistance against insect herbivores.
Often working synergistically, ethylene and jasmonate signaling leads to increased plant resistance [105].Early genes encoding enzymes in ethylene biosynthesis, AtACS2, AtACS8 and AtACS4, as well as the transcription factors AtRAP2.6,AtERF2 and AtERF114, were wound-induced in WT or both genotypes 1 h post-damage (Figure 5A,C).

Signal Transduction
In this and other studies, AtMAPKKK17, induced late in response to wounding in WT plants (Pattern 5, Figure 5E), activates the MKK3-MPK1/2/7 module [114,115].In plants challenged with the two-spotted spider mite, Tetranchus urtica, plants with higher constitutive AtMAPKKK17 expression had higher resistance against mite herbivory, exhibiting less leaf damage with mites having lower fecundity [58].AtMAPKKK21 is expressed early after wounding in both genotypes (Pattern 1, Figure 5A).In contrast to MAPKKK17, this kinase was found to be a negative regulator of plant resistance to mites [58].

Physical Defense
Foliar trichomes create a mechanical defense against insect herbivores and, as well, may be a site for the production of chemical defenses [116].Even though arabidopsis trichomes are non-glandular, volatile organic compounds as well as aliphatic and indolic GSLs may be biosynthesized in these cells [21,50,117].In addition, arabidopsis trichomes may serve as mechanosensors, possibly leading to specialized metabolite production when triggered [118,119].AtGL3, a late wound-induced gene that shows higher stressassociated expression in WT compared with lox2 plants (Pattern 5, Figure 5E), encodes a bHLH transcription factor that regulates trichome initiation as well as anthocyanin biosynthesis [120,121].Constitutively, this transcription factor interacts with JAZ proteins, specifically, JAZ1, JAZ2, JAZ8 and JAZ11, leading to its repression [122].In response to wounding, the resultant degradation of JAZ proteins coupled to increased AtGL3 expression supports observations of a wound-associated increase in arabidopsis foliar trichomes [123].

Chemical Defense
GSLs and flavonoids are important defensive compounds in the Brassicaceae [18,124,125].A wound-induced difference in foliar GSL levels was not observed (Figure 7).However, in both genotypes, a strong expression of GSL biosynthetic genes was observed late after wounding, which corresponds to the light phase in our experiment.This supports previous observations of a possible role of circadian rhythms in foliar GSL biosynthesis [21].
In contrast, a genotype difference in wound-induced genes associated with flavonoid biosynthesis was identified in this study, with genes expressed more highly in WT plants (Pattern 5, Figure 6).Flavonoids can act as feeding or oviposition deterrents or toxins against insect herbivores [124,125].Even though AtRNS1, a jasmonate-independent, woundinduced gene that encodes a ribonuclease that is a negative regulator of anthocyanin biosynthesis [126], is expressed in both genotypes (Pattern 4, Figure 5D), the late woundinduced expression of genes that encode transcriptional regulators or flavonoid biosynthetic enzymes was observed in WT plants (Pattern 5, Figure 5E).Thus, particularly in WT plants, there is a strong upregulation of genes involved in flavonoid biosynthesis, particularly anthocyanins, that may act as antioxidants or be involved in plant resistance against insect herbivory [125,127].

Indirect Chemical Defense
Wounding of arabidopsis leaves results in the biosynthesis and release of volatile organic compounds (VOCs) that are involved in numerous ecological roles, including intra-plant stress signaling and tritrophic interactions, such as attracting parasitoids or predators of the herbivore [128,129].From the diterpenoid precursor geranylgeranyl diphosphate, AtTPS04, which is expressed in arabidopsis non-glandular trichomes [50], catalyzes the production of (E,E)-geranyllinalool, which is converted by AtCYP82G1 into volatile 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) [49], an important attractant in tritrophic interactions for beneficial parasitoid and predatory arthropods [130].AtTPS04 is induced in response to herbivory by caterpillars of the specialist diamond backmoth, Plutella xylostella, or the generalist African cotton leafworm, Spodoptera littoralis [131][132][133][134].In our study, both AtTPS04 and AtCYP82G1 show early wound-induced gene expression (Pattern 1, Figure 5A).AtCYP82G1 is also induced in response to the treatment of arabidopsis leaves with spider mite egg extract [113].
AtHPL had higher late wound-induced expression in WT compared with lox2 plants (Pattern 5, Figure 5E).This jasmonate-responsive gene is also induced in response to herbivory by P. rapae or S. littoralis caterpillars [141].Cytosolic AtHPL competes with the jasmonate biosynthetic pathway for 13-hydroxyperoxide precursors to generate alkenals, which are converted into C6 volatiles [83].The AtHPL in the arabidopsis Col-0 ecotype (which was also used in this study) has a deletion resulting in a truncated protein that is unable to use 13-hydroperoxide linolenic acid as a precursor but uses 13-hydroperoxide linoleic acid to produce hexenals [142].
AtBSMT1 was also more highly wound-induced in WT plants (Pattern 2, Figure 5B).This methyltransferase, involved in volatile methyl salicylate biosynthesis, is induced in response to P. rapae and Pieris brassicae herbivory [143,144].The resultant methyl salicylate is an attractant for female parasitic Diadegma semiclausum wasps and also deters oviposition by female P. brassicae butterflies, thus decreasing herbivore damage [144,145].
In addition to the role that these VOCs play in attracting natural enemies of the herbivore [146], these volatiles can also act on the plant itself, leading to systemic upregulation of plant defense responses [147,148].Recently, the importance of these volatiles as conspecific signals has been recognized [149].Thus, volatiles may serve as systemic signals resulting in the induction of plant defense responses.

Interference with Insect Nutrition or Physiology
Obtaining sufficient nitrogen to maintain development and fitness is a key challenge for phytophagous insects [150].Thus, plants have numerous strategies to interfere with a herbivore's ability to obtain sufficient nitrogen to limit herbivore success.
In herbivorous insects, serine proteinases, such as trypsin and chymotrypsin, initiate protein digestion [151]; However, plants produce inhibitors of these enzymes, known as proteinase inhibitors [152].Overexpression of a soybean trypsin inhibitor in arabidopsis negatively affected the larval biomass of corn earworm, Helicoverpa zea, caterpillars [153].In our study, we identified the expression of three wound-induced trypsin inhibitors.AtTI1 is induced early in both genotypes and has previously been found to be induced by aphid attack (Pattern 1, Figure 5A) [154].AtKTI3 and AtTI6 are more highly expressed in WT plants late after wounding (Pattern 5, Figure 5E).
Another late wound-induced gene identified in our transcriptomic study that interferes with nitrogen resources for the insect encodes arginase (AtARGAH2) (Pattern 5, Figure 5E).This enzyme catabolizes nitrogen-rich arginine to produce urea and ornithine.Direct feeding of Manduca sexta caterpillars on tomato plants overexpressing arginase resulted in smaller insects, presumably because of the decreased availability of the essential amino acid arginine [86].However, this may also reflect the potential toxicity of arginase products, where ornithine may be converted by AtNATA1, a late pattern 4 gene (Figure 5D), to N d -acetylornithine [155].This derivative negatively affects the fecundity of the green peach aphid, Myzus persicae.It is of interest that the pH optimum of plant arginase is alkaline [86], which likely allows it to be most active in its native cellular location, the mitochondrial matrix [156], and also in the alkaline midgut of caterpillars [157].In addition to these activities, ARGAH2 activity may affect nitric oxide (NO) production [158].Though this has been shown in the marine green algae Ostreococcus tauri [159], in higher plants, NOS-like activity is controversial; however, NO has been proposed to be released from arginine upon its conversion to citrulline [160,161].Consistent with this, AtARGAH2 knockout plants had increased NO accumulation [158].Differences in NO, or its more biologically stable form Snitrosoglutathione (GSNO), can impact protein S-nitrosation and S-glutathionylation status of regulatory proteins involved in plant defense [162][163][164][165]. Manduca sexta caterpillars grew larger on plants silenced in their ability to produce GSNO.Methyl jasmonate-induced levels of proteinase inhibitors and some defensive specialized metabolites (caffeoylputrescine, diterpene glycosides) were lower in these plants [162].Therefore, AtARGAH2 may affect caterpillar nutrition or plant defense.
The phloem-associated protein AtPP2-A5 has two domains, a PP2 (lectin activity) domain at the C-terminus and a Toll/Interleukin-1 receptor domain at the N-terminus [166].Though this protein plays a major role in sealing wounded sieve elements (Pattern 1, Figure 5A), AtPP2-A5 overexpression or knockout lines show modified transcriptional patterns to spider mite herbivory, suggesting that the receptor portion of this protein recognizes an herbivore-specific effector to remodel gene expression [166].The knockout line was more susceptible to spider mite damage, and the arthropods had higher mortality on the overexpression lines.This likely reflects direct interactions with the insect, potentially by binding to the arthropod gut epithelial [167].Feeding aphids a diet spiked with recombinant AtPP2-A5 did not affect mortality but did negatively affect the weight gain of two different aphid species, the pea aphid Acythosiphon pisum (~30% smaller) and the green peach aphid Myzus persicae (10-20% smaller) [168].Aphids reared on AtPP2-A5 overexpression lines had lower colonization and spent less time feeding on the phloem compared with WT plants [169].
Chlorophyllase, AtCHL1, catalyzes the hydrolysis of chlorophyll to chlorophyllide.Under stress conditions, this enzyme may be involved in chlorophyll degradation to minimize chlorophyll-associated ROS generation [170,171].However, as this protein is associated with the endoplasmic reticulum or tonoplast, only upon cell disruption, such as that incurred by herbivory, does the enzyme come into contact with its substrate, chlorophyll.The product of this reaction, chlorophyllide, is toxic to S. littoralis caterpillars and binds to the midgut of Bombyx mori caterpillars, potentially impairing digestion [172].However, other studies suggest that chlorophyllide may have roles that benefit the insect herbivore [173].In our study, AtChl1 was induced late after wounding and showed higher expression in WT plants (Pattern 5, Figure 5E).

Plant Maintenance
Arabidopsis thaliana wildtype (WT) Columbia (Col-0) seeds were obtained from the Arabidopsis Biological Resource Center.Seeds of the lox2-1 mutant line, which has a mutation in the tryptophan amino acid at position 630 to produce a stop codon resulting in a truncated non-functional protein, were generously provided by Dr. E. E. Farmer [28].The seeds were surface-sterilized in 70% (v/v) ethanol for one min, then 0.6% (v/v) bleach (NaOCl) for 3 min, followed by five successive washes in sterile ddH 2 O. Between these treatments, the seeds were recovered by centrifugation, and the liquid was removed.The seeds were then placed in Petri dishes containing Murashige and Skoog salts, pH 5.8, in 0.8% agar, followed by stratification in the dark at 4 • C to promote synchronized germination.After 2 days, the Petri dishes were transferred to a growth cabinet with 14 h of light with an intensity of 250 µmol m −2 s −1 followed by 10 h darkness.The temperature of the light-dark cycle was 23:20 • C.After one week, the germinated seedlings were transplanted into pots (12 cm diameter × 11 cm height) containing Fafard Agromix G6 potting medium and grown under the same conditions.Plants were bottom-watered 3 times per week with 20:20:20 NPK fertilizer (0.14 g/L distilled water) and used for experimentation at the vegetative 3.9 stage [174].

Experimental Design
Two days prior to the wounding experiment, a plexiglass sheet was placed between randomly chosen plants that were to remain unwounded or mechanically damaged to separate the treatments and avoid volatile signaling between these groups.At 9 PM (time 0; dark phase), arabidopsis lines were either mechanically damaged, whereby each leaf of the rosette was wounded once by a hole punch, without harming the mid-vein, or left unwounded.Whole rosettes were collected for phytohormones (1 h; dark phase), GSLs (12 h; light phase) or transcriptomics (1 h and 12 h, dark and light phase, respectively) post-damage, flash-frozen in liquid nitrogen and stored at −80 • C until analysis.The experiment was repeated temporally three times to collect samples for transcriptomics and five times for phytohormone and GSL analyses.At each temporal replicate, one sample was taken for the different analyses (RNA-Seq or metabolite analysis) of transcriptomics (n = 3), phytohormones (n = 5) and GSLs (n = 5).

Phytohormone Analysis
Following the protocol described in Martinez Henao et al. [175], plant samples were finely ground and extracted in ethyl acetate containing isotopically labeled standards (D6-JA, D6-JA-Ile and D4-SA (OlChemim, s.r.o)).The samples were vigorously vortexed and centrifuged (19,000× g, 10 min, 4 • C), and the supernatant was transferred to a new tube.The extraction was repeated, and the supernatants were pooled.Following evaporation using a vacuum concentrator at room temperature, the resulting pellet was resuspended in 70% (v/v) methanol (HPLC-MS grade).A final centrifugation step was performed as above to ensure the removal of all non-soluble debris.Metabolites were separated by ultrahigh performance liquid chromatography (UHPLC) followed by detection on a triple quadrupole mass spectrometer (EVOQ-TQ-MS, Bruker, Hamburg, Germany).Reverse phase UHPLC was performed using a Zorbax Extend-C18 column (4.6 × 50 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA, USA).The mobile phase began with 5% (v/v) acetonitrile (ACN), 0.05% (v/v) formic acid for 30 s and then increased to 50% (v/v) ACN, 0.05% (v/v) formic acid over 2 min.After separation, the compounds were nebulized by electron spray ionization and detected using the EVOQ-TQ-MS.Phytohormones were identified based on their retention time, in comparison with known standards, as well as their m/z.

Glucosinolate Analysis
GSLs were extracted from lyophilized leaf tissue and analyzed by high-performance liquid chromatography (HPLC)-pulsed amperometric detection following Grosser and van Dam [176].Briefly, 70% MeOH was added to the finely ground tissues and incubated at 90 • C for 6 min to inactivate myrosinases followed by sonication for 15 min.After centrifugation (2975× g for 10 min), the supernatant was transferred to a clean tube, and the pellet was re-extracted.Pooled supernatants were passed through a diethylaminoethyl Sephadex A-25 ion exchange column preconditioned with ddH 2 O.After washing (2 × 1 mL 70% MeOH, 2 × 1 mL ddH 2 O, 1 × 1 mL 20 mM sodium acetate buffer, pH 5.5), the column was treated with 10 U of arylsulfatase and incubated for 12 h at RT. Desulfated GSLs were eluted in sterile MilliQ H 2 O (2 × 0.75 mL) and lyophilized.

RNA Extraction, Library Preparation and Transcriptomics
Total RNA was extracted from flash-frozen pulverized plant rosettes (~100 mg) using the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) following the manufacturer's protocol with an additional on-column DNase digestion step to avoid genomic DNA contamination.The 2100 Bioanalyzer instrument (Agilent Technologies) was used to determine RNA quality using an RNA integrity number (RIN) of 8.7 as a minimum threshold.Total RNA samples were processed by Genome Québec Innovation Centre for library preparation and next-generation RNA sequencing (RNA-Seq).Libraries were generated from 250 ng of total RNA as follows: mRNA enrichment and ribosomal RNA removal were performed using the Next Poly(A) Magnetic Isolation following the manufacturer's instructions (New England BioLabs, Ipswich, USA).cDNA synthesis was performed with Next RNA First Strand Synthesis and Next Ultra Directional RNA Second Strand Synthesis kits (New England BioLabs).The remaining steps of library preparation were performed using the Next Ultra II DNA Library Prep Kit for Illumina (New England BioLabs).Adapters and PCR primers were purchased from New England BioLabs.
The libraries were normalized, pooled and denatured in 0.02 N NaOH, followed by neutralization using HT1 hybridization buffer.Twenty-four strand-specific mRNA libraries were generated and sequenced by loading 200 pM on the NovaSeq 6000 S4 (Illumina) using the Xp protocol as per the manufacturer's recommendations, with paired-end (PE) mode for 2 × 100 base pairs (bp) resulting in >25 million reads in each direction.The Illumina phiX control v3 library was used as a control and mixed with libraries at a 1% level.Base calling was performed using Real-Time Analysis (RTA) v3 software.The program bcl2fastq2 v2.20 was used to demultiplex samples and generate fastq reads.

Quality Check, Clipping and Mapping
Raw read quality was examined by performing FastQC 0.11.9 on ".fastq.gz"file [177].FastQC produces a control statistic and evaluates every metric using a classification system of pass, warn or fail.The adapter clipping and bad end quality trimming were performed by subjecting the raw reads to fastp tool version 0.20.1 to remove adapters, low-quality bases with a Phred score less than 20 and reads shorter than 25 bp from the tail (3' end) from further analysis [178].This preprocessing was followed by another quality check with FastQC.Read mapping was conducted using a splice-aware alignment tool STAR v2.7.9a to align the trimmed reads against the arabidopsis reference genome downloaded from The Arabidopsis Information Resource (TAIR, assembly ID: TAIR10, https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR10_gff3/TAIR10_GFF3_genes.gff (accessed on 9 December 2022)) [179].

Differential Gene Expression
Strand-specific transcript abundance was calculated using featureCounts (a tool included in the subread package 2.0.3) on mapped and sorted BAM files.The resulting read counts per transcript values were imported into the web-based tool NetworkAnalyst 3.0 for data filtering, Log 2 normalization and differential analysis [180].The statistical method DESeq2 with a negative binomial (Gamma-Poisson) distribution was used to identify differentially expressed genes using the log 2 -fold change (logFC) calculation [181,182].Genes with a False Discovery Rate (FDR)-corrected p-value (padj) ≤ 0.05 and log 2 fold change ≥ 2 or ≤−2 were deemed significant.The transcriptomic data generated by this study are available in the Supplementary Materials of this article (Supplemental Table S1) and the raw read data (FASTQ) were deposited in the NCBI Sequence Read Archive (Bioproject ID #PRJNA1077722).

Statistics
For phytohormone and GSL analyses, a two-factor analysis of variance (ANOVA) (factors: genotype and treatment) was conducted using the statistical program SPSS vers.29 (Supplemental Tables S2 and S3).

Conclusions
By comparing early (1 h) and late (12 h) constitutive and wound responses between arabidopsis WT and lox2 mutants, we identified gene expression differences related to the regulation of jasmonate biosynthesis and, in mechanically damaged plants, resistance to insect herbivores.Both constitutively and in wounded plants, SA levels are higher in lox2, which is reflected in the expression of SA-responsive genes.Since jasmonate and SA pathways are mutually antagonistic [106], this suggests that AtLOX2 may be involved in the constitutive biosynthesis of jasmonates that modulate SA levels.
As expected, the wound-associated jasmonate burst is dampened in lox2 compared with WT plants.In the early response to wounding, genes are similarly expressed in both genotypes with few genotype-specific differences.In contrast, in the later transcriptional responses, higher expression of numerous genes involved in insect resistance is observed in WT plants compared to lox2, highlighting the role of AtLOX2 in arabidopsis resistance to insects (Figure 8).  .General wound-induced responses found in both genotypes.Jasmonate biosynthesis begins in the chloroplast from 18C membrane lipidderived precursors, typically α-linolenic acid, to finally form one of the biologically active jasmonates, jasmonoyl-isoleucine (JA-Ile).Numerous genes encoding enzymes in the jasmonate biosynthetic pathway are upregulated in wounded plants.JA-Ile enters the nucleus and forms a bridge between the SCF COI1 and jasmonate ZIM domain (JAZ) repressors, leading to their degradation through the proteasome.The degradation of JAZ proteins releases MYC2/3/4 transcription factors, leading to jasmonate-responsive gene expression.The abscisic acid (ABA)-PYL6 receptor complex positively interacts with MYC2.Genes encoding JAZ-negative regulators as well as those further metabolizing jasmonic acid (JA) (i.e., JOX, ST2A) to inactive derivatives are also wound-induced.Gibberellin (GA) bound to its receptor GID1 activates a pathway that leads to the proteasome-mediated degradation of negative DELLA growth regulators, such as RGL3.In wounded leaves, the increase in RGL3 expression and genes that encode GA2OX6 and GA2OX8, involved in gibberellin metabolism to inactive products, results in the suppression of plant growth.Numerous genes involved in oxidative stress are wound-induced.For example, the oxidative stress-associated transcription factor RRTF1 and MDAR3, which are part of the Foyer-Halliwell-Asada cycle, a series of interconnected enzymatic reactions to detoxify the reactive oxygen species hydrogen peroxide (H 2 O 2 ).Wound-induced MYB15 leads to the expression of ELI3/CAD8 and PRX52, which contribute to lignin biosynthesis.TPS04 and CYP82G1 are involved in the biosynthesis of volatiles, such as 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), that are attractive to natural enemies of the herbivorous insect.TI1 and NATA1 contribute to plant resistance against arthropods, whereas MAPKKK21 is a negative regulator of arthropod resistance.(B).LOX2-specific responses.Wound-induced genes expressed at higher levels in WT compared with lox2 plants.Wound-induced expression of genes that encode LOX2 and MYC2 involved in jasmonate biosynthesis and signaling, respectively, is noted.CORI3 is a cysteine lyase involved in cysteine biosynthesis that produces precursors for ethylene, aliphatic glucosinolates (GSLs) and reduced glutathione (GSH) involved in the Foyer-Halliwell-Asada cycle.Also, the expression of the gene encoding DHAR1 in this pathway is wound-induced.Other cellular antioxidants whose biosynthetic pathway is positively regulated in these plants are anthocyanins.Indirect defenses involved in volatile biosynthesis are wound-induced.An increase the genes that encode proteins involved in antinutritive defenses, such as CLH1, ARGAH2 and TIs, as well as MAPKKK17, an important signaling kinase involved in arthropod resistance, is also seen.Abbreviations: ABA: abscisic acid, AOC: allene oxide cyclase, AOS: allene oxide synthase, DHAR1: dehydroascorbate reductase1, GA: gibberellin, GSH: reduced glutathione, GSL: glucosinolate, GSSG: oxidized glutathione, JA: jasmonic acid, JA-Ile: jasmonoyl-isoleucine, JAZ: jasmonate-Zim domain, JOX: jasmonate oxidase, LOX: lipoxygenase, OPDA: 12-oxo-phytodienoic acid, OPR: oxo-phytodienoate reductase, OPCL: OPC-8-CoA ligase, PUFA: polyunsaturated fatty acid, TF: transcription factor, TMTT: 4,8,12-trimethyltrideca-1,3,7,11-tetraene.

Data Availability Statement:
The data that support the findings of this study are available within this article.The transcriptomic data (FASTQ) generated by this study are available through the NCBI Sequence Read Archive (Bioproject ID #PRJNA1077722).

Figure 1 .Figure 1 .
Figure 1.Foliar constitutive gene expression.(A): Venn diagram of constitutive genes differentially expressed between wildtype (WT) and lox2 Arabidopsis thaliana plants at two time points (1 and 12 h).Differentially expressed genes between the different treatments and times were determined by DESeq2 (p-value (padj) ≤ 0.05 and log2 fold change ≥2 or ≤−2).(B): Gene set enrichment analysis (GSEA) and ridgeline plot highlighting constitutive metabolic pathways differentially expressed between WT and lox2 plants at 1 h.(C): GSEA and ridgeline plot highlighting constitutive metabolic pathways differentially expressed between WT and lox2 plants at 12 h.The ridgeline plot visualizes the distribution of differential enrichment categories identified by GSEA.Significantly differentially expressed pathways in ridgeline plots are indicated in green.

Figure 4 .
Figure 4. Wound-induced foliar gene expression.Four-week-old Arabidopsis thaliana wildtype (WT) or lox2 plants were wounded with a hole punch on each fully expanded rosette leaf and harvested at 1 and 12 h.Gene set enrichment analyses (GSEA) and ridgeline plots of WT (1 h) (A,E), WT (12 h) (B,F), lox2 (1 h) (C,G) and lox2 (12 h) (D,H).Wound-induced differentially expressed genes (DEGs) were determined by DESeq2 (p-value (padj) ≤ 0.05 and log2 fold change ≥2 or ≤−2).The ridgeline plot visualizes the distribution of differential enrichment categories identified by GSEA.Significantly differentially expressed pathways in ridgeline plots are indicated in green.In general, WT and lox2 plants showed similar wound-induced responses (Figure

Figure 4 .
Figure 4. Wound-induced foliar gene expression.Four-week-old Arabidopsis thaliana wildtype (WT) or lox2 plants were wounded with a hole punch on each fully expanded rosette leaf and harvested at 1 and 12 h.Gene set enrichment analyses (GSEA) and ridgeline plots of WT (1 h) (A,E), WT (12 h)

Figure 6 .
Figure 6.Wound-induced genes involved in polyphenol biosynthesis.Four-week-old Arabidopsis thaliana wildtype (WT) or lox2 plants were wounded with a hole punch on each fully expanded rosette leaf and harvested at 1 or 12 h.(A) Transcriptional regulators and (B) biosynthetic genes.Wound-induced genes were determined by DESeq2 (p-value (padj) ≤ 0.05 and log2 fold change ≥2

Figure 7 .
Figure 7. Foliar glucosinolate (GSL) levels and expression of GSL biosynthesis genes.Four-weekold Arabidopsis thaliana wildtype (WT) or lox2 plants were wounded with a hole punch on each fully

Figure 7 .
Figure 7. Foliar glucosinolate (GSL) levels and expression of GSL biosynthesis genes.Four-week-old Arabidopsis thaliana wildtype (WT) or lox2 plants were wounded with a hole punch on each fully

Figure 8 .
Figure 8. Wound-induced genes involved in plant-insect interactions in arabidopsis WT and lox2 mutants.Foliar gene expression in wounded lox2 mutants, which has a truncated non-functional enzyme, was compared to WT plants.Proteins encoded by early genes (1 h) are shown in blue, whereas later transcript expression (12 h) is depicted in brown.(A).General wound-induced responses found in both genotypes.Jasmonate biosynthesis begins in the chloroplast from 18C membrane lipid-derived precursors, typically α-linolenic acid, to finally form one of the biologically active jasmonates, jasmonoyl-isoleucine (JA-Ile).Numerous genes encoding enzymes in the

Figure 8 .
Figure 8. Wound-induced genes involved in plant-insect interactions in arabidopsis WT and lox2 mutants.Foliar gene expression in wounded lox2 mutants, which has a truncated non-functional enzyme, was compared to WT plants.Proteins encoded by early genes (1 h) are shown in blue, whereas later transcript expression (12 h) is depicted in brown.(A).General wound-induced responses found in both genotypes.Jasmonate biosynthesis begins in the chloroplast from 18C membrane lipidderived precursors, typically α-linolenic acid, to finally form one of the biologically active jasmonates, jasmonoyl-isoleucine (JA-Ile).Numerous genes encoding enzymes in the jasmonate biosynthetic pathway are upregulated in wounded plants.JA-Ile enters the nucleus and forms a bridge between the SCF COI1 and jasmonate ZIM domain (JAZ) repressors, leading to their degradation through the

Author Contributions:
The experimental design was conceived by D.K. and J.C.B. and conducted by D.K. (wounding experiments, transcriptomic analyses), J.M.H. (transcriptomic analyses) and C.L. (molecular training) under the supervision of J.R. and J.C.B.The phytohormone analysis was conducted by A.S. under the supervision of N.M.v.D. The data were analyzed and this manuscript was written by D.K. and J.C.B., with valuable input from J.M.H., C.L., A.S., N.M.v.D. and J.R. All authors have read and agreed to the published version of the manuscript.Funding: We gratefully acknowledge our funding sources for supporting this research including the Deutsche Forshungsgemeinshaft (DFG-FZT 118, 202548816) (NMvD, AS), the Natural Science and Engineering Research Council (NSERC) Discovery grants (NSERC-2019-04516 to JCB and NSERC-2019-05955 to JR), and the Fonds de recherche du Québec-nature et technologies (FRQNT) Centre SÈVE (FRQNT-RQT00335) to JCB and JR.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.